scholarly journals Simple and Fast DNA Based Sensor System for Screening of Small-Molecule Compounds Targeting Eukaryotic Topoisomerase 1

Pharmaceutics ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1255
Author(s):  
Kamilla Vandsø Petersen ◽  
Asier Selas ◽  
Kirstine Mejlstrup Hymøller ◽  
Karol Mizielinski ◽  
Maria Thorsager ◽  
...  
Keyword(s):  
Small Molecule ◽  
Rolling Circle Amplification ◽  
Amplification Product ◽  
Rolling Circle ◽  
Topoisomerase 1 ◽  
Potential Target ◽  
Enzyme Screening ◽  
Anti Cancer ◽  
The Individual ◽  
Cancer Activity

Background: Eukaryotic topoisomerase 1 is a potential target of anti-parasitic and anti-cancer drugs. Parasites require topoisomerase 1 activity for survival and, consequently, compounds that inhibit topoisomerase 1 activity may be of interest. All effective topoisomerase 1 drugs with anti-cancer activity act by inhibiting the ligation reaction of the enzyme. Screening for topoisomerase 1 targeting drugs, therefore, should involve the possibility of dissecting which step of topoisomerase 1 activity is affected. Methods: Here we present a novel DNA-based assay that allows for screening of the effect of small-molecule compounds targeting the binding/cleavage or the ligation steps of topoisomerase 1 catalysis. This novel assay is based on the detection of a rolling circle amplification product generated from a DNA circle resulting from topoisomerase 1 activity. Results: We show that the binding/cleavage and ligation reactions of topoisomerase 1 can be investigated separately in the presented assay termed REEAD (C|L) and demonstrate that the assay can be used to investigate, which of the individual steps of topoisomerase 1 catalysis are affected by small-molecule compounds. The assay is gel-free and the results can be detected by a simple colorimetric readout method using silver-on-gold precipitation rendering large equipment unnecessary. Conclusion: REEAD (C|L) allows for easy and quantitative investigations of topoisomerase 1 targeting compounds and can be performed in non-specialized laboratories.

scholarly journals Cyanine dye mediated mitochondrial targeting enhances the anti-cancer activity of small-molecule cargoes

2020 ◽  
Vol 56 (34) ◽  
pp. 4672-4675 ◽  
Author(s):  
Alexander R. Nödling ◽  
Emily M. Mills ◽  
Xuefei Li ◽  
Davide Cardella ◽  
Edward J. Sayers ◽  
...  
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Cyanine Dye ◽  
Mitochondrial Targeting ◽  
Anti Cancer ◽  
Cancer Activity

Conjugation of small molecules to a simple cyanine dye can lead to organelle-specific delivery.

Mo1929 Small-Molecule Parkinson's Disease Kinase Inhibitor LRRK2-in-1 Demonstrates Potent Anti-Cancer Activity Through Inhibition of DCLK1

2014 ◽  
Vol 146 (5) ◽  
pp. S-694
Author(s):  
Nathaniel Weygant ◽  
Dongfeng Qu ◽  
William L. Berry ◽  
Randal May ◽  
Parthasarathy Chandrakesan ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
Anti Cancer ◽  
Cancer Activity

scholarly journals Direct detection of mRNA expression in microbial cells by fluorescence in situ hybridization using RNase H-assisted rolling circle amplification

2019 ◽  
Author(s):  
Hirokazu Takahashi ◽  
Kyohei Horio ◽  
Setsu Kato ◽  
Toshiro Kobori ◽  
Kenshi Watanabe ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Direct Detection ◽  
Rolling Circle Amplification ◽  
Rnase H ◽  
Microbial Cells ◽  
Rolling Circle ◽  
The Individual ◽  
Meta Analyses

ABSTRACTMeta-analyses using next generation sequencing is a powerful strategy for studying microbiota; however, it cannot clarify the role of individual microbes within microbiota. To know which cell expresses what gene is important for elucidation of the individual cell’s function in microbiota. In this report, we developed novel fluorescence in situ hybridization (FISH) procedure using RNase-H-assisted rolling circle amplification to visualize mRNA of interest in microbial cells without reverse transcription. Our results show that this method is applicable to both gram-negative and gram-positive microbes without any noise from DNA, and it is possible to visualize the target mRNA expression directly at the single-cell level. Therefore, our procedure, when combined with data of meta-analyses, can help to understand the role of individual microbes in the microbiota.

scholarly journals High-throughput, cost-effective verification of structural DNA assembly

2013 ◽  
Vol 42 (4) ◽  
pp. e22-e22 ◽  
Author(s):  
Yandi Dharmadi ◽  
Kedar Patel ◽  
Elaine Shapland ◽  
Daniel Hollis ◽  
Todd Slaby ◽  
...  
Keyword(s):  
High Throughput ◽  
Low Cost ◽  
Rolling Circle Amplification ◽  
Cost Effective ◽  
Building Blocks ◽  
Pathway Engineering ◽  
Dna Assembly ◽  
Rolling Circle ◽  
Enzyme Screening ◽  
First Time

Abstract DNA ‘assembly’ from ‘building blocks’ remains a cornerstone in synthetic biology, whether it be for gene synthesis (∼1 kb), pathway engineering (∼10 kb) or synthetic genomes (>100 kb). Despite numerous advances in the techniques used for DNA assembly, verification of the assembly is still a necessity, which becomes cost-prohibitive and a logistical challenge with increasing scale. Here we describe for the first time a comprehensive, high-throughput solution for structural DNA assembly verification by restriction digest using exhaustive in silico enzyme screening, rolling circle amplification of plasmid DNA, capillary electrophoresis and automated digest pattern recognition. This low-cost and robust methodology has been successfully used to screen over 31 000 clones of DNA constructs at <$1 per sample.

scholarly journals Reconstitution of Authentic Nanovirus from Multiple Cloned DNAs

2009 ◽  
Vol 83 (20) ◽  
pp. 10778-10787 ◽  
Author(s):  
Ioana Grigoras ◽  
Tatiana Timchenko ◽  
Lina Katul ◽  
Ana Grande-Pérez ◽  
Heinrich-Josef Vetten ◽  
...  
Keyword(s):  
Faba Bean ◽  
Acyrthosiphon Pisum ◽  
Rolling Circle Amplification ◽  
Virus Genome ◽  
Biological Properties ◽  
Rolling Circle ◽  
Consensus Sequences ◽  
Circular Dnas ◽  
The Individual ◽  
Dna Components

ABSTRACT We describe a new plant single-stranded DNA (ssDNA) virus, a nanovirus isolate originating from the faba bean in Ethiopia. We applied rolling circle amplification (RCA) to extensively copy the individual circular DNAs of the nanovirus genome. By sequence analyses of more than 208 individually cloned genome components, we obtained a representative sample of eight polymorphic swarms of circular DNAs, each about 1 kb in size. From these heterogeneous DNA populations after RCA, we inferred consensus sequences of the eight DNA components of the virus genome. Based on the distinctive molecular and biological properties of the virus, we propose to consider it a new species of the genus Nanovirus and to name it faba bean necrotic stunt virus (FBNSV). Selecting a representative clone of each of the eight DNAs for transfer by T-DNA plasmids of Agrobacterium tumefaciens into Vicia faba plants, we elicited the development of the typical FBNSV disease symptoms. Moreover, we showed that the virus thus produced was readily transmitted by two different aphid vector species, Aphis craccivora and Acyrthosiphon pisum. This represents the first reconstitution of a fully infectious and sustainably insect-transmissible nanovirus from its cloned DNAs and provides compelling evidence that the genome of a legume-infecting nanovirus is typically comprised of eight distinct DNA components.

scholarly journals Organometallic Small Molecule Kinase Inhibitors – Direct Incorporation of Re and 99mTc into Opaganib®

2021 ◽  
Author(s):  
Roger Alberto ◽  
Raphael Lengacher ◽  
Youchao Wang ◽  
Henrik Braband ◽  
Olivier Blacque ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Small Molecule ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Ic50 Value ◽  
Anti Cancer ◽  
Direct Incorporation ◽  
Cancer Activity

[(η5-Cp)ReI(CO)3] was incorporated into the kinase inhibitor Opaganib®. The resulting bioorganometallic complex showed a similar anti-cancer activity to Opaganib® against PC-3 cancer cells. The IC50 value for the kinase SK2...

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